Hsp104 is a protein chaperone that helps cells recover from stress by resolubilizing proteins from aggregates. This disaggregation activity requires assistance of Hsp40 and Hsp70 and is necessary for fragmentation of prion fibers that underlies replication of amyloid-based yeast prions. Depleting or inactivating Hsp104 arrests prion replication, which causes yeast prions to be lost as cells divide because the number of cells eventually becomes greater than the number of infectious prion particles, or seeds. Elevating expression of Hsp104 under conditions where it is not normally induced also leads to prion loss. The mechanism of this curing is unknown, but differences in curing kinetics, dependency on other factors, and the inadequacy of Hsp104 disaggregation activity alone suggest it is unrelated to that of curing by Hsp104 inactivation. To assess more definitively how overexpressing Hsp104 cures cells of prions, we monitored the fate of prion particles (fluorescent foci) in live cells using a Sup35-GFP fusion protein after overexpressing Hsp104. We found a large heterogeneity in the distribution of fluorescent foci in cells of partially cured populations and distinct kinetics of prion loss that were incompatible with the mechanism being inhibition of fragmentation. We also found no differences in segregation pattern of prion seeds, ruling out asymmetric segregation of prion seeds as a possible mechanism. Our data support a mechanism by which prions are removed in a two-step process of trimming by Hsp104 to reduce their size followed by elimination, probably by proteolysis. Expressing Hsp104 is being explored as a therapeutic approach in mammalian amyloid models. Our work is providing insight into the mechanisms underlying functions of this chaperone system that might reveal unanticipated limitations or benefits of such an approach.